Molecular diagnosis and clinical features of 206 patients with 46, XY disorders of sexual development / 中华内分泌代谢杂志

Objective:To investigate methods of molecular diagnosis and clinical features of 46, XY disorders of sexual development(DSD).Methods:A total of 206 cases of 46, XY DSD patients, who visited the Shanghai Ninth People′s Hospital, Shanghai Jiaotong University School of Medicine, from July 2009 to June 2021, underwent AA chip based on multiplex PCR and probe-capture-targeted next-generation sequencing. Clinical features of patients with genetic diagnosis were analyzed.Results:Among 206 patients, the diagnostic rate of patients with micropenis, hypospadias and cryptorchidism was the highest, up to 75.28%. Almost all patients had different degrees of undermasculinized external genitalia. The most frequent phenotype was micropenis with hypospadias(87.25%). Only one gene variant was detected in 81 patients(39.32%), multiple genetic variants were detected in 104 patients(50.49%), and no gene variant was identified in 21 patients(10.19%). 107 patients had definite genetic diagnosis, with a diagnostic rate of 51.94% by adding the pathogenic and likely pathogenic ratios following the American College of Medical Genetics and Genomics(ACMG) guidelines, including 40 patients of steroid 5α-reductase type 2(SRD5A2) variants(37.38%), 36 patients of androgen receptor(AR) variants(33.64%), 13 patients of steroidogenic factor 1(NR5A1) variants(16.82%), 6 patients of 17β-hydroxysteroid dehydrogenases 3(HSD17B3) variants(5.61%), 2 patients of 17α-hydroxylase/17, 20-lyase enzyme(CYP17A1), Wilms′ tumor 1(WT1) and GATA binding protein 4(GATA4) variants(1.87%), and one patient of luteinizing hormone receptor(LHCGR) variant(0.93%). Gynecomastia was found in 29 of 81 postpubertal patients, of which 25(86.21%) had AR variants.Conclusions:46, XY DSD presents complex clinical manifestations and molecular etiologies. Targeted nextgeneration sequencing has the advantages of high throughput, high efficiency and low cost, which has a high value especially in etiological diagnosis of 46, XY DSD with large genetic heterogeneity.

Standardization of an organic DNA extraction method from dried blood spots and its downstream molecular applications in neonatal screening and diagnostic confirmation of lysosomal disorders

Introduction: Dried blood spot (DBS) samples have been used for diagnostic purposes since their introduction in the neonatal screening of phenylketonuria almost 50 years ago. The range of its application has been extended to modern approaches, such as next-generation sequencing (NGS) for molecular genetic testing. This study aimed to evaluate the use of a standardized organic method for DNA extraction from DBS samples in the diagnostic setting.Methods: The clinical applicability of the method was tested using 3 samples collected from a newborn screening project for lysosomal storage diseases, allowing the determination of the genotype of the individuals. DNA was extracted from 3 3-mm diameter DBS punches. Quality, purity, and concentration were determined, and method performance was assessed by standard polymerase chain reaction, restriction length polymorphism, Sanger sequencing, and targeted NGS.Results: Results were compared with the ones obtained from DNA samples extracted following the internally validated in-house extraction protocol that used 6 3-mm punches of DBS and samples extracted from whole blood.Conclusion: This organic method proved to be effective in obtaining high-quality DNA from DBS, being compatible with several downstream molecular applications, in addition to having a lower cost per sample

Prevalence of gene mutations in a Chinese 46,XY disorders of sex development cohort detected by targeted next-generation sequencing / 亚洲男科学杂志(英文版)

46,XY disorders of sex development (DSD) is characterized by incomplete masculinization genitalia, with gonadal dysplasia and with/without the presence of Müllerian structures. At least 30 genes related to 46,XY DSD have been found. However, the clinical phenotypes of patients with different gene mutations overlap, and accurate diagnosis relies on gene sequencing technology. Therefore, this study aims to determine the prevalence of pathogenic mutations in a Chinese cohort with 46,XY DSD by the targeted next-generation sequencing (NGS) technology. Eighty-seven 46,XY DSD patients were enrolled from the Peking Union Medical College Hospital (Beijing, China). A total of fifty-four rare variants were identified in 60 patients with 46,XY DSD. The incidence of these rare variants was approximately 69.0% (60/87). Twenty-five novel variants and 29 reported variants were identified. Based on the American College of Medical Genetics and Genomics (ACMG) guidelines, thirty-three variants were classified as pathogenic or likely pathogenic variants and 21 variants were assessed as variants of uncertain significance. The overall diagnostic rate was about 42.5% based on the pathogenic and likely pathogenic variants. Androgen receptor (AR), steroid 5-alpha-reductase 2 (SRD5A2) and nuclear receptor subfamily 5 Group A member 1 (NR5A1) gene variants were identified in 21, 13 and 13 patients, respectively. The incidence of these three gene variants was about 78.3% (47/60) in patients with rare variants. It is concluded that targeted NGS is an effective method to detect pathogenic mutations in 46,XY DSD patients and AR, SRD5A2, and NR5A1 genes were the most common pathogenic genes in our cohort.

Implementation of a Targeted Next-Generation Sequencing Panel for Constitutional Newborn Screening in High-Risk Neonates

PURPOSE: Newborn screening (NBS) programs are important for appropriate management of susceptible neonates to prevent serious clinical problems. Neonates admitted to neonatal intensive care units (NICU) are at a potentially high risk of false-positive results, and repetitive NBS after total parenteral nutrition is completely off results in delayed diagnosis. Here, we present the usefulness of a targeted next-generation sequencing (TNGS) panel to complement NBS for early diagnosis in high-risk neonates. MATERIALS AND METHODS: The TNGS panel covered 198 genes associated with actionable genetic and metabolic diseases that are typically included in NBS programs in Korea using tandem mass spectrometry. The panel was applied to 48 infants admitted to the NICU of Severance Children's Hospital between May 2017 and September 2017. The infants were not selected for suspected metabolic disorders. RESULTS: A total of 13 variants classified as likely pathogenic or pathogenic were detected in 11 (22.9%) neonates, including six genes (DHCR7, PCBD1, GAA, ALDOB, ATP7B, and GBA) associated with metabolic diseases not covered in NBS. One of the 48 infants was diagnosed with an isobutyl-CoA dehydrogenase deficiency, and false positive results of tandem mass screening were confirmed in two infants using the TNGS panel. CONCLUSION: The implementation of TNGS in conjunction with conventional NBS can allow for better management of and earlier diagnosis in susceptible infants, thus preventing the development of critical conditions in these sick infants.

Identification of a Novel Homozygous Splice-Site Mutation in that Causes Progressive Myoclonus Epilepsy with or without Renal Failure / 中华医学杂志(英文版)

<p><b>Background</b>Progressive myoclonus epilepsies (PMEs) comprise a group of rare genetic disorders characterized by action myoclonus, epileptic seizures, and ataxia with progressive neurologic decline. Due to clinical and genetic heterogeneity of PMEs, it is difficult to decide which genes are affected. The aim of this study was to report an action myoclonus with or without renal failure syndrome (EPM4) family and summarize the clinical and genetic characteristics of all reported EPM4 patients.</p><p><b>Methods</b>In the present study, targeted next-generation sequencing (NGS) was applied to screen causative genes in a Chinese PME family. The candidate variant was further confirmed by cosegregation analysis and further functional analysis, including the reverse transcription polymerase chain reaction and Western blot of the proband's muscle. Moreover, literature data on the clinical and mutational features of all reported EPM4 patients were reviewed.</p><p><b>Results</b>The gene analysis revealed a novel homozygous splicing mutation (c.995-1G>A) of the SCARB2 gene in two brothers. Further functional analysis revealed that this mutation led to loss function of the SCARB2 protein. The classification of the candidate variant, according to the American College of Medical Genetics and Genomics standards and guidelines and functional analysis, was pathogenic. Therefore, these two brothers were finally diagnostically confirmed as EPM4.</p><p><b>Conclusions</b>These present results suggest the potential for targeted NGS to conduct a more rapid and precise diagnosis for PME patients. A literature review revealed that mutations in the different functional domains of SCARB2 appear to be associated with the phenotype of EPM4.</p>

Clinical, pathological, imaging and genetic analysis of two cases of central core disease with different inheritance modes / 中国神经精神疾病杂志

Objective To study the clinical, pathological, imaging features of two cases of central core disease (CCD) with different inheritance and to explore the similarities and differences between autosomal recessive CCD (AR-CCD) and autosomal dominant CCD (AD-CCD). Methods Clinical manifestations, family history, muscle MRI and muscle biopsy were collected. Targeted next generation sequencing (NGS) and sanger sequencing were applied for genetic analysis. Co-segregation analysis was further conducted in one family. Results Their common clinical manifestations included childhood early-onset proximal limbs muscle weakness and dystrophy accompanied with facial involvement. The MRI revealed extensive muscular dystrophy and fatty filtration in the both thighs, but not in rectus femoris. Pathology of skeletal muscle showed typical central cores in type Ⅰ muscle fibers and eccentric cores only in AR-CCD. Targeted NGS identified 3 missense mutations in RYR1, including one novel mutation. Conclusion The present study has described clinical and pathological features of two typical CCD patients with different inheritance, which may be associated with the different mutations in RYR1 gene. Targeted NGS apparently improves the genetic diagnosis of CCD.